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mRNA-Lipoplex Loaded Microbubble Contrast Agents for Ultrasound-Assisted

Transfection of Dendritic Cells

Marie-Luce De Temmermana,§, Heleen Dewittea,§, Roosmarijn E. Vandenbrouckeb,c, Bart

Lucasa, Claude Libertb,c, Jo Demeestera, Stefaan C. De Smedta,*, Ine Lentackera,† and

Joanna Rejmana,†,*

§Both authors contributed equally to this work

†Both senior authors contributed equally to this work

aLaboratory of General Biochemistry and Physical Pharmacy, Ghent Research Group on

Nanomedicine, Faculty of Pharmaceutical Sciences, Ghent University, Harelbekestraat

72, Ghent, B-9000 Belgium

bDepartment for Molecular Biomedical Research, VIB, Technologiepark 927, 9052

Ghent, B-9052 Belgium

cMolecular Mouse Genetics Unit, Department of Biomedical Molecular Biology, Ghent

University, Technologiepark 927, Ghent, B-9052 Belgium

*Corresponding authors: Tel +32 9 264 80 76; Fax +32 9 264 81 89; Email addresses:

Stefaan.DeSmedt@UGent.be (S.C. De Smedt), j_rejman@hotmail.com (J. Rejman)

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ABSTRACT

In cancer immunotherapy the immune system should be triggered to specifically

recognize and eliminate tumor cells in the patient’s body. This could be achieved by

loading dendritic cells (DCs) with tumor associated antigens (TAAs). This can be

achieved by transfecting DCs with messenger RNA encoding a tumor-associated

antigen. Here we demonstrate transient transfection of dendritic cells by means of

mRNA lipoplexes bound to microbubbles. Microbubble-attached lipoplexes were

introduced into the cells by applying ultrasound. Our data demonstrate that ultrasound-

mediated delivery of mRNA complexes led to efficient transfection of DCs. When mRNA

encoding luciferase was used, maximal levels of the enzyme activity were detected 8h

after ultrasound application. Upon longer incubation protein expression gradually

declined. This treatment did not affect viability of the cells. Intracellular localisation of

mRNA-lipoplexes in DCs was determined by flow cytometry using fluorescently labelled

lipoplexes. Over 50 % of DCs contained fluorescently labelled mRNA-complexes. In the

absence of additional maturation signals, transfection of immature DCs with mRNA-

lipoplex loaded microbubbles and ultrasound application induced only a minor shift in the

expression level of maturation markers (CD40 and CD86). However, in the presence of

the activation stimulus (LPS), cells were able to further mature as shown by a significant

up-regulation of CD40 expression. Thus, our results demonstrate that mRNA-lipoplex

loaded microbubbles can serve as an applicable and safe tool for efficient mRNA-

transfection of cultured DCs.

KEYWORDS: mRNA, microsphere, gene therapy, controlled drug release

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INTRODUCTION

Increasing knowledge of immunology and biology of dendritic cells (DCs) has

sparked interest in immunotherapy as a strategy for the treatment of cancer. DCs are

regarded as the most potent antigen presenting cells (APCs) that are able to initiate and

regulate T and B cell-mediated immunity [1]. Immature DCs function as sentinels

throughout the body where they recognize and capture antigens. These antigens are

subsequently processed and DCs migrate to the lymph nodes. On their way, they

mature from antigen-sampling cells into APCs. In the lymph nodes, they present the

antigen-derived peptides associated with MHC class I and II molecules to naive T cells,

thereby inducing an immune response [2]. For several reasons, vaccination strategies

based on modification of DCs to present tumor associated antigens (TAAs) offer an

attractive approach to stimulate the immune system of a patient to achieve therapeutic

immunity against tumors [3]. First of all, DCs are expected to give rise to tumor-specific

T cells that induce tumor regression or eradication. Moreover, DCs generate tumor-

specific memory cells that might help to prevent tumor relapse [4-6]. Although preclinical

and clinical studies demonstrated encouraging priming of T cells, clinical responses are

so far mainly limited to disease stabilization and prolonged survival. Complete remission

was only seen in low percentage of treated patients [7]. Therefore, the development of

novel vaccination strategies still remains a key issue.

In recent years, the focus of research has shifted from peptide and protein

loading of DCs towards genetic approaches, where plasmid DNA (pDNA) or messenger

RNA (mRNA) is delivered into isolated DCs [8-10]. Translation of the proteins by the DC

itself is attractive as it paves the way to antigen presentation not only by MHC class II

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but also via MHC class I molecules involved in the generation of cellular immune

responses. Moreover, these strategies hold the potential to introduce multiple epitopes,

ultimately yielding an immune response against the broad antigenic repertoire of the

tumor. As compared to DNA-based approaches, mRNA-based vaccination presents

several advantages. Firstly, mRNA does not integrate into the genome, rendering its

application much safer. Secondly, while in non-dividing cells, the nuclear envelope

represents a serious obstacle for the delivery of pDNA into the nucleus, this obviously

does not apply to mRNA [11]. Finally, although mRNA generates only transient protein

expression, this should be adequate for antigen processing and presentation by DCs.

To deliver mRNA to DCs, numerous strategies have been explored. Despite the

appealing results obtained with viral vectors based on adenovirus, poxvirus and

lentivirus [12, 13], there are some considerable disadvantages associated with virus-

based approaches. Their application has been restricted due to toxicity and the

immunogenicity of the vector itself [14]. Moreover, the production of viral vectors is

costly and laborious and the size of the carried transgene is limited [15]. As a result,

non-viral approaches represent a promising alternative to deliver genetic material. At

present, electroporation is most frequently used to deliver mRNA to DCs [16-18]. The

transient permeabilization of the plasma membrane caused by the applied electric fields

allows direct entry of the mRNA into the cytoplasm, hence potentially leading to

immediate high protein expression. However, several aspects limit the practical

applicability of this strategy. It has only a limited range of applications in vivo as the

target tissue must be easily accessible (skin or muscle). As a consequence, in the

context of cancer immunotherapy, DCs have to be isolated from the patient’s blood and

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subsequently transfected ex vivo. Apart from being expensive and laborious, this

approach has to deal with several additional issues such as the yield of DCs after

aphaeresis, the cell culture and freezing procedures and, ultimately, the re-

administration to the patient. In order to circumvent these drawbacks it would be

desirable to develop novel approaches which could potentially be used to deliver mRNA

to DCs in vivo.

In this study, we developed and characterized ultrasound-responsive

microbubbles loaded with mRNA-lipoplexes as an alternative physical method to deliver

mRNA to the cytosol of DCs. Apart from their clinical use as ultrasound contrast agents,

microbubbles have been investigated also as drug and gene delivery vehicles [19, 20].

They consist of a gas core surrounded by a stabilizing lipid or polymer layer. In response

to ultrasound waves, the microbubbles cavitate, and eventually implode [21]. Cavitation

and especially implosion is known to transiently open nearby cells by forming temporary

pores in their plasma membranes, which allows direct entry of larger molecules such as

nucleic acids into the cytosol, thus bypassing endocytic pathways [22]. Using an

ultrasound-based approach for DC transfection is particularly appealing as ultrasound is

a widely applied medical imaging and therapy tool, appreciated for its non-invasive and

safe character.

The specific aims of this study were (a) to evaluate the potential of ultrasound-

responsive microbubbles loaded with mRNA-complexes to transfect DCs, (b) to

measure the levels and kinetics of marker protein production following transfection, and

(c) to reveal the impact of ultrasound treatment on the viability and maturation status of

DCs.

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MATERIALS AND METHODS

Cell culture

DC primary cultures were generated from bone marrow of C57Bl/6 mice. Female

C57BL/6 mice were purchased from Janvier (Le Genest Saint Isle, France) and housed

in a specified pathogen-free facility according to the regulations of the Belgian law and

the local Ethical Committee. Mice were sacrificed and bone marrow was flushed out of

the femur and tibia. After red blood cell lysis (Pharm Lyse Buffer, BD Biosciences), cells

were seeded at a cell density of 5x105 cells/ml in OptiCellsTM (Nunc, Thermo Scientific,

Aalst, Belgium) and incubated at 37°C in 5 % CO2. The cell culture medium was RPMI-

1640 (Gibco-Invitrogen, Merelbeke, Belgium) supplemented with 5 % FCS (Hyclone,

Pierce, Rockford, IL, USA), 1 % penicillin / streptomycin (Gibco-Invitrogen, Merelbeke,

Belgium), 1 % L-glutamine (Gibco-Invitrogen, Merelbeke, Belgium) and 50 µM β-

mercaptoethanol (Gibco-Invitrogen, Merelbeke, Belgium) and contained also 10 ng/ml

IL-4 (Peprotech, Rock Hill, NJ) and 10 ng/ml GM-CSF (Peprotech, Rock Hill, NJ). At day

2 and 6 of culture, the non-adherent cells were collected by centrifugation, resuspended

in fresh culture medium and seeded to the same OptiCellTM. The cells were treated 7

days after seeding.

Messenger RNA

To produce mRNA by in vitro transcription, plasmids encoding luciferase and

green fluorescent protein (EGFP) were purified using a QIAquick PCR purification kit

(Qiagen) and linearized using Dra I restriction enzyme (plasmid encoding firefly

luciferase) or Spe I (plasmid encoding EGFP). Linearized plasmids were used as

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templates for the in vitro transcription reaction using the T7 mMessage mMachine kit

(Ambion). mRNAs were purified by DNase I digestion and precipitated with LiCl. This

was followed by washing with 70 % ethanol. The produced mRNAs were both capped

and polyadenylated. The mRNA concentration was determined by measuring the

absorbance at 260 nm. mRNAs were stored in small aliquots at -80°C at a concentration

of 1 µg/µl.

Bioluminescence assay

All cells were removed from OptiCellsTM and collected by centrifugation. After

removing the culture medium, the cells were washed once with PBS (Gibco/Invitrogen,

Merelbeke, Belgium). Subsequently, 100 µl of Cell Culture Lysis Reagent (Promega,

Leiden, The Netherlands) was added. After incubation for 30 min, the samples were

centrifuged (12,000 rpm at 4°C for 5 min) and 40 µl aliquots of the supernatants were

transferred to a 96-well plate. Luciferase activity of each sample was assayed in a

GloMaxTM 96 Luminometer (Promega, Leiden, The Netherlands). 100 µl of the substrate

solution was added to each well and the emitted light was measured over a 10 s period.

A standard Bradford assay was employed to determine the protein content of each

sample (Biorad, Nazareth Eke, Belgium). The results are expressed as relative light

units (RLU) per milligram of protein.

Lipoplexes

If not indicated differently, liposomes were composed of 42.5 % DOTAP (1,2-

dioleoyl-3-trimethylammonium-propane), 42.5 % DOPE (1,2-dioleoyl-sn-glycero-3-

phosphoethanolamine) and 15 % DSPE-PEG-2000-biotin [1,2-distearoyl-sn-glycero-3-

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phosphoethanol-amine-N-[biotinyl(polyethylene glycol)-2000] (all from Avanti Polar

Lipids, Alabaster, AL). This was done by transferring the appropriate amounts of lipids,

dissolved in CHCl3 into a round-bottom flask. The CHCl3 was evaporated under nitrogen

and the resulting lipid film was rehydrated in RNase-free water (Ambion, Lennik,

Belgium). The resulting DOTAP/DOPE/PEG liposomes were sonicated. The total lipid

concentration in these liposomes was 1 mg/ml. Fluorescently labeled liposomes were

obtained by incorporation of 1 % NBD-PE (phosphatidylethanolamine-N-(7-nitro-1,3-

benzoxadiol-4-yl)) (Avanti Polar Lipids, Alabaster, AL).

Preparation and characterization of biotinylated microbubbles

Microbubbles were prepared starting from a solution of a mixture of DPPC (1,2-

dipalmitoyl-sn-glycero-3-phosphocholine) (Lipoid, Ludwigshafen, Germany) and DSPE-

PEG-biotin [1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotin(polyethylene

glycol)-2000] (Avanti Polar Lipids, Alabaster, AL) in a 1:2:7 glycerol-propyleneglycol-H2O

solvent (Sigma-Aldrich, Bornem, Belgium); the molar ratio of the lipids in the lipid

solutions was 85:15. The lipid solution was prepared as follows. Appropriate aliquots of

both lipids, dissolved in CHCl3, were transferred to a round bottom flask. After CHCl3

evaporation, the lipid film was dissolved in a 1:2:7 glycerol-propyleneglycol-H2O mixture

to obtain a clear solution with a final lipid concentration of 4.6x10-4 mmol/ml. Aliquots of

this lipid solution were transferred to 2.5 ml chromatography vials, the headspace of

which was filled with C4F10 gas (F2 chemicals, Preston, UK). Finally, microbubbles were

obtained by high-frequency shaking of the lipid solution in a Capmix™ device (3M-

ESPE, Diegem, Belgium) during 15 s. The size and the concentration of the

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microbubbles in the dispersion (i.e. number of microbubbles per ml) were determined

with a Beckman-coulter Multisizer 4 (Beckman-coulter, Brea, CA).

Transfection

mRNA encoding luciferase or EGFP was complexed with cationic liposomes and

the resulting complexes were attached to the microbubble surface via avidin-biotin

interaction, as shown in Fig. 2A. The cells were transfected 7 days after seeding.

Different amounts of DOTAP/DOPE/PEG liposomes were dispersed in 50 µl of OptiMem

(Invitrogen, Merelbeke, Belgium). This solution was mixed with 50 µl of mRNA solution

in OptiMem. After 10 min of incubation at RT, 900 µl of OptiMem was added.

Subsequently, the mixture was added to the microbubble solution in OptiMem. After a

short incubation of the cells with microbubbles loaded with mRNA-complexes,

ultrasound was applied (Sonitron 2000, Artison, Inola, OK, USA) (1 MHz, 2 W/cm², 50 %

duty cycle, 30 s).

BM-DC staining for flow cytometric analysis and confocal microscopy

DCs were identified using anti-CD11c surface stain. Transfected cells were

stained for surface CD11c following the manufacturer’s protocols and instructions (BD

Pharmingen, Erembodegem, Belgium). Briefly, cells were surface stained for CD11c-

APC or CD11c-FITC (BD Pharmingen) in stain buffer (BD Pharmingen) for 1 h at 4°C.

Cells were washed twice and resuspended in flow buffer (PBS containing 1 % BSA and

0,1 % azide) for flow cytometric analysis or confocal microscopy.

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Dendritic cell activation

The effect of the mRNA-lipoplex loaded microbubbles and ultrasound application

on the DC maturation status was assessed by evaluating up-regulation of the maturation

surface markers CD40 and CD86. Briefly, following transfection the cells were

transferred to 24 well plates and cultured with or without addition of E. coli derived

lipopolysaccharide (LPS, 1 µg/ml, Sigma Aldrich, Bornem, Belgium) to the culture

medium. 24 h after exposure of DCs to mRNA-lipoplexes, microbubbles and/or

ultrasound, the cells were washed with stain buffer (BD Pharmingen) and incubated on

ice for 30 min with 5 % goat serum in PBS. Then cells were stained for CD11c-FITC in

combination with staining for CD40-PE or CD86-PE (all BD Pharmingen) for 1 h on ice,

followed by washings with stain buffer. Untreated and LPS stimulated cells were used as

negative and positive controls respectively. All samples were analyzed on a BD flow

cytometer.

FACS analysis

FACS data were acquired using a FACSCaliburTM flow cytometer (BD

Pharmingen) and data were analyzed using CellQuestTM software. In all experiments,

DCs were identified based on their CD11c-APC or CD11c-FITC surface staining.

Intracellular localization of mRNA-lipoplexes was determined by using fluorescently

labeled liposomes. Transfection efficiency was evaluated by measuring EGFP

expression in gated DCs.

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Confocal Microscopy

Confocal microscopy images of mRNA-lipoplex loaded microbubbles and

transfected DCs were recorded using a Nikon C1si confocal laser scanning module

attached to a motorized Nikon TE2000-E inverted microscope (Nikon Benelux, Brussels,

Belgium). A sample of microbubbles or cell suspension was placed on a cover glass and

analyzed with CLSM using a water immersion objective lens (Plan Apo 60X, NA 1.2,

collar rim correction, Nikon).

Toxicity assay

Cytotoxicity was evaluated 24 h after transfection of DCs in OptiCellsTM using a

SYTOX® green nucleic acid stain (Molecular Probes/Invitrogen, Merelbeke, Belgium).

24 h after treatment, the cells were washed in HEPES buffer and nuclei of non-viable

cells were stained with a 45 nM SYTOX® green solution for 30 min on ice. Prior to flow

cytometric analysis, samples were diluted in flow buffer.

RESULTS

Characterization of microbubbles loaded with mRNA-lipoplexes

To avoid degradation by nucleases and to achieve sustained circulation time in

vivo nucleic acids-based drugs are often complexed with cationic lipids and modified

with polyethylene glycol (PEG). The latter also serves to prevent complex clearance by

the reticulo-endothelial system [23] thus enhancing the chance of uptake by the target

cells.

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As described in the material and methods section, complexes were prepared by

mixing mRNA with DOTAP/DOPE liposomes. Lipoplexes thus formed were attached to

microbubbles via biotin-avidin-biotin bridges as schematically depicted in Fig. 2A. To be

responsive to clinically applied ultrasound frequencies (1-5 MHz) and enable

microbubble implosion, microbubbles should ideally have a diameter between 1 and

7 µm [24]. We determined the size of microbubbles by coulter counter measurements.

The mean volume diameter of microbubbles employed in this study was 4.4 ± 1.6 µm

(consistent with a mean number diameter of 2.3 ± 1.2 µm), which meets clinical

requirements. The effective binding of mRNA-complexes to microbubbles was

demonstrated by confocal microscopy. As shown in Fig. 2B fluorescently labelled

mRNA-lipoplexes clearly mark the bubble surface.

Ultrasound-triggered delivery of mRNA-lipoplexes

Intracellular localisation of mRNA-lipoplexes in DCs was determined by flow

cytometry using fluorescently labelled lipoplexes. As shown in Fig. 3 over 50 % of DCs

contained fluorescently labelled mRNA-complexes. By contrast, negligible uptake of

complexes (< 1 %) was observed if cells were incubated with free mRNA-lipoplexes or

lipoplex-loaded microbubbles without application of ultrasound.

The insignificant uptake of free mRNA-complexes by DCs can be explained by

the high degree of PEGylation (15-20 %) of the complexes used in this study. It is known

that such a hydrophilic coating reduces uptake of the complexes because it shields their

positive charge thereby preventing interactions with the negatively charged cell surface

[25]. Moreover, it has been demonstrated that PEGylated particles, taken up by the cells

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by endocytosis, get trapped in the endo-lysosomal compartment, which impedes their

release into the cytosol [26]. One possible way to overcome this problem is entry of

PEGylated particles directly into the cytosol as achieved by ultrasound-assisted delivery.

Our group has shown that microbubble implosion creates temporary cell membrane

perforations and at the same time pushes the complexes through such pores [27].

Transfection efficiency of mRNA-lipoplex loaded microbubbles

To assess the ability of mRNA-lipoplex loaded microbubbles to transfect DCs, an

mRNA encoding luciferase was employed. The expression kinetics was characterized by

determining the levels of luciferase at different time points. For this reason we used

firefly luciferase mRNA as this is known to have a very rapid intracellular turnover with a

half-life of 3 h [28]. As shown in Fig. 4, significant protein expression occurs already as

early as 30 min after mRNA-delivery. Maximal levels of luciferase were detected 8 h

after ultrasound application. Upon longer incubation, protein levels gradually declined.

Furthermore, no luciferase activity was detected when only mRNA-lipoplexes were

added to the cells, consistent with the insignificant uptake of mRNA-complexes by the

DCs if neither microbubbles nor ultrasound were applied. This proves that mRNA

transfection of dendritic cells can indeed be triggered by means of ultrasound and thus

opens possibilities towards an image-guided spatiotemporally controlled vaccination

system, as will be discussed later on.

In addition to measuring total levels of protein expression, we evaluated the

number of transfected cells. To that end DCs were transfected with complexes carrying

mEGFP. The proportion of DCs expressing EGFP was evaluated 24 h after transfection

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by means of flow cytometry. The influence of the transfection protocol on the

background fluorescence signal of the DCs was verified by using mRNA encoding

luciferase. Application of ultrasound in combination with mRNA-lipoplex loaded

microbubbles resulted in transfection up to 24 % of DCs (Fig. 5A). Ultrasound-assisted

transfection of DCs with free mRNA-lipoplexes resulted in very low numbers of

transfected cells (< 1 %; data not shown). These results were confirmed by confocal

analysis of transfected DCs. Fig. 5B shows surface-stained DCs (anti-CD11c-APC)

expressing EGFP.

These transfection data provide proof of concept for the use of mRNA-lipoplex

loaded microbubbles to transfect DCs. Although 50 % of DCs were shown to contain

mRNA-complexes after this treatment (Fig. 3), only 24 % of the cells expressed EGFP

(Fig. 5A). This may be due to the incomplete release of the mRNA from the complexes.

mRNA electroporation has been shown to achieve transfection efficiencies of up

to 70 % for DCs [18]. However, Grünebach et al. proved that high transfection

efficiencies are not essential to elicit effective T cell responses. They even succeeded in

eliciting antigen-specific cytotoxic T cell (CTL) responses with low to hardly detectable

(< 1 %) transfection efficiencies [17]. Several suggestions explaining this phenomenon

were brought forward. Firstly, the CTL assy is possibly more sensitive than flow

cytometry in detecting low levels of antigen. Secondly, not all antigenic peptides that are

recognized by CTLs are necessarily derived from biologically functional proteins. Even

protein fragments translated from damaged mRNA could be processed into antigenic

peptides that can elicit CD8 positive T cells. Taking all this together, we may conclude

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that the transfection results obtained with mRNA-lipoplex loaded microbubbles and

ultrasound are likely to be sufficient for effective stimulation of the immune system.

Cell viability of DCs following ultrasound exposure

The potential of mRNA delivery by means of ultrasound can be properly

evaluated only if potentially toxic effects on the cells are taken into consideration. To

assess the effect of the mRNA-complexes, microbubbles and ultrasound on DC viability,

we utilized a SYTOX® green nucleic acid stain. Flow cytometric analysis indicated a

survival of 93 % of the cells after exposure to mRNA-lipoplex loaded microbubbles, 91 %

after exposure to unloaded microbubbles combined with ultrasound and 84 % after

addition of mRNA-lipoplex loaded microbubbles with ultrasound application (Fig. 6). This

corresponds with data reported by Suzuki et al. and indicates that most of the dendritic

cells are indeed able to completely restore the ultrasound-induced cell membrane

damage within 24 h [29]. The percentage of dead cells after sonoporation is comparable

to that resulting from electroporation, where 80-90 % viability has been reported for

human monocyte-derived DCs, depending on the physical parameters used [16, 17].

Moreover, the 91 % toxicity observed when unloaded bubbles were used in combination

with ultrasound correlates with prior results of our group on other cell types [30]. This

indicates that DCs are not particularly sensitive to sonoporation-induced damage.

Maturation status of transfected DCs

In the context of DC-based vaccination strategies the DC maturation status

should be taken into consideration. This is a series of changes in the DC phenotype,

shifting DC function from antigen-sampling to antigen-presentation. As a result, this

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maturation process is crucial for the ability of DCs to prime naive T cells [31, 32]. We

determined whether the mRNA-lipoplex loaded microbubbles and the ultrasound

treatment have an influence on DC maturation. To that end, DCs were evaluated with

respect to the expression of the co-stimulatory markers (CD40 and CD86) 24 h after

transfection. Moreover, we investigated the susceptibility of treated DCs to additional

maturation stimuli. Therefore, DCs were cultured in the absence or presence of a

maturation stimulus (lipopolysaccharide, LPS) added immediately after transfection.

An overview of the phenotypic maturation of DCs in response to mRNA-lipoplex

loaded microbubbles and ultrasound is given in Fig. 7. In the absence of additional

maturation signals, transfection of immature DCs with mRNA-lipoplex loaded

microbubbles and ultrasound application induced only a minor shift in the expression

level of CD40 and a slightly higher increase of CD86, compared to that in the untreated

cells. However, in the presence of the activation stimulus LPS, cells were able to further

mature as shown by a significant up-regulation of CD40 expression. We take this to

indicate that the transfection procedure did not negatively affect the capacity of DCs to

respond to pathogen-associated molecular patterns (PAMPs) and to become activated.

Immature DCs exposed to either ultrasound or microbubbles did not express higher

levels of CD40 and CD86, while LPS stimulation of these cells induced expression of

these markers. Taken together, these observations indicate that the slight phenotypical

changes in DC maturation status were solely due to the mRNA-complexes which had

been delivered and that the transfection procedure itself did not induce phenotypical

changes in the treated DCs.

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DISCUSSION

We were able to show that mRNA-lipoplexes can be attached to the surface of

ultrasound-responsive microbubbles via biotin-avidin-biotin bridges. Secondly, we

demonstrated an effective uptake of mRNA-lipoplexes by DCs resulting in a significant

expression of luciferase and EGFP by DCs after ultrasound-guided transfection with

mRNA-lipoplex loaded microbubbles, without compromising DC viability and maturation

capacities. In 2009 Suzuki and colleagues reported on the use of microbubbles and

ultrasound to pulse DCs with ovalbumine [29]. They showed that this resulted in MHC I

presentation and the development of a strong CD8+ T cell response. These results are

very encouraging as this indicates that the combination of microbubbles and ultrasound

can be a powerful vaccination tool. We believe that, compared to the system proposed

by Suzuki et al. [29] for vaccination purposes, the development of an mRNA-lipoplex

loaded microbubble offers several advantages. Firstly, when considering the use of

autologous tumor-derived antigens, even small tumor samples may yield sufficient

amounts of mRNA, while for protein extraction relatively large tumor samples are

required. Moreover, co-transfection with mRNA encoding maturation stimuli is possible,

leading to enhanced cytokine signaling, thus strengthening the desired immune

response [33].

The most important outcome of our findings is that ultrasound-assisted

transfection of DCs might eliminate the need of ex vivo procedures in the development

of a DC vaccine, as we will elaborate below. This is important because besides the

problems of costs and labor-intensiveness of current DC vaccination procedures, it has

been suggested that the capacity of the in vitro cultured and transfected DCs to migrate

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to the lymph nodes is sub-optimal [34]. To address this drawback, the DC vaccine has

been injected intranodally [35, 36]. Alternatively, in vivo transfection of DCs could be

considered. The latter is particularly interesting, consindering that currently used ex vivo

generated monocyte-derived DCs are subject to debate. It has become more and more

clear that this type of DCs merely represents one of the many subsets that can be found

in vivo. As it remains unclear which particular DC subtypes are responsible for the most

efficient CTL priming, it would be advantageous to transfect several of these DC

subtypes in an in vivo setting [32, 37]. For this purpose, the ideal location would be the

lymph nodes, as these organs are populated by large numbers of both resident and

migratory DCs. In this connection, it has been reported that intranodal delivery of naked

pDNA resulted in an important increase of vaccine immunogenicity [38, 39]. Although

this is a promising observation, the unprotected DNA is rapidly cleared and still has to

make its way across the plasma membrane, avoid intralysosomal degradation and cross

the barrier of the nuclear membrane before it can be transcribed and translated into an

antigenic protein. Therefore, this approach is obviously amenable to substantial

improvement. Microbubbles and ultrasound could provide a possible solution, allowing

an increase in transfection efficiency of intranodally located DCs. We care to stress here

that besides the in vitro proof presented in the present work, satisfactory transfection

efficiencies with microbubbles and ultrasound have been shown also in vivo [40, 41].

Furthermore, our suggestion to use microbubbles for intranodal DC transfection is

supported by the consideration that microbubbles have already been used clinically as

ultrasound contrast agents in the lymphatics, and more specifically, as means to

visualize sentinel lymph node metastases [42, 43]. This would imply that a non-invasive

intradermal injection of the mRNA-lipoplex loaded microbubbles would be sufficient to

19

obtain intranodal localization of the nucleic acids. Intranodal location of the microbubbles

– and hence of the mRNA – can easily be visualized as the presence of the bubbles will

be revealed by enhanced ultrasound contrast. This way, the complete vaccination

procedure could be divided into a first phase of low intensity ultrasound, allowing

microbubble visualization and a second phase, where the ultrasound intensity is

increased to cause microbubble collapse and subsequent DC transfection.

CONCLUSIONS

Our results demonstrate that mRNA-lipoplex loaded microbubbles can be used as an

efficient ultrasound-triggered transfection tool for DCs, without compromising cell

viability or DC maturation capacities. These findings are especially important in the

context of a possible in vivo use of this technique. Needless to say that direct in vivo

transfection of DCs would not only eliminate the need for costly ex vivo DC handling and

transfection, but it could also allow transfection of several DC subsets which would

broaden the generated immune response.

ACKNOWLEDGEMENTS

Heleen Dewitte is a doctoral fellow of the Institute for the Promotion of Innovation

through Science and Technology in Flanders, Belgium (IWT-Vlaanderen). Ine Lentacker

is a postdoctoral fellow of the Research Foundation-Flanders, Belgium (FWO-

Vlaanderen). The support of both these institutions is gratefully acknowledged. We

would also like to thank this institution for granting the BRAINSTIM project. This

research was funded through the FWO research Grant G.0187.11.

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[FIGURES]

Figure 1.

Figure 2.

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Figure 3.

Figure 4.

24

Figure 5.

Figure 6.

25

Figure 7.

[FIGURE CAPTATIONS]

Figure 1. Schematic representation of ultrasound mediated mRNA-lipoplex delivery into the cytosol of a DC.

Upon mRNA translation, antigenic protein is produced, followed by the processing of antigen into peptide fragments

which can be loaded onto MHC molecules.

Figure 2. mRNA-lipoplex-loaded microbubbles. (A) Schematic representation of an mRNA-lipoplex-loaded

microbubble. To generate mRNA-lipoplex loaded microbubbles, mRNA was first complexed with cationic liposomes

consisting of DOTAP, DOPE and DSPE-PEG-biotin. The resulting mRNA complexes were loaded onto the

microbubble surface via avidin-biotin interaction. (B) Confocal micrographs of avidinylated microbubbles after

incubation with biotinylated fluorescently labelled mRNA-lipoplexes: (1) overlay; (2) fluorescently labelled lipoplexes;

(3) transmission.

26

Figure 3. Intracellular localisation of fluorescently labelled mRNA lipoplexes. Fluorescently labeled lipoplexes,

containing 1 % NBD-PE, were attached to the microbubbles and added to DCs. 15 min after ultrasound exposure, the

cells were thoroughly washed to remove non-internalized complexes, which was followed by anti-CD11c-APC surface

staining and flow cytometric analysis. Gated DCs were further analysed.

Figure 4. Transfection efficiency of mRNA lipoplex-loaded microbubbles. mRNA encoding luciferase was

complexed with DOTAP/DOPE/PEG lipoplexes and added to microbubbles and ultrasound was applied. Levels of

luciferase activity in DCs at different time points after ultrasound exposure were assayed by measuring

bioluminescence. Graphs represent means ± standard deviation (n=6). Transfection efficiency of free PEGylated

lipoplexes was assayed two hours after adding them to the cells.

Figure 5. Transfection efficiency of mRNA lipoplex-loaded microbubbles. (A) mRNA encoding GFP was

complexed with DOTAP/DOPE/PEG liposomes (15 % PEG – formulation 1; 20 % PEG – formulation 2) and added to

microbubbles and ultrasound was applied. Control cells were transfected with mRNA encoding luciferase. DCs were

analyzed for GFP expression 24 h after transfection by flow cytometry. DCs were gated based on CD11c-APC

staining. (B) Confocal images of DCs after transfection with mRNA lipoplex-loaded microbubbles and ultrasound. (1)

overlay; (2) CD11c stain; (3) EGFP expression; (4) transmission).

Figure 6. Cell viability. DCs were exposed to mRNA-lipoplex loaded microbubbles or microbubbles and ultrasound

or mRNA-lipoplex loaded microbubbles and ultrasound. Following the treatment, cells were stained with SYTOX®

green nucleic acid stain to identify dead cells. Viability of untreated cells was set as 100 %. Results are the mean ±

standard deviation (n=3).

Figure 7. Maturation of DCs. Representative histograms illustrating phenotypic maturation of DCs in response to

mRNA-lipoplex loaded microbubbles and ultrasound. 24 h after transfection DCs, cultured with or without LPS, were

stained for CD40 or CD86 maturation markers and assessed by flow cytometry. DCs were gated on CD11c. Untreated

DCs and LPS-stimulated DCs served as negative and positive controls, respectively.